In vitro antioxidant and hepatoprotective activity of isolated compounds from Pistacia integerrima.Introduction
The majority of diseases are linked to oxidative stress due to free radicals. Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and metabolism. Free radicals contribute to more than one hundred diseases including arthritis, ischemia, ageing and liver disorders (Squadriato 1998). Plants and plant products have been used as sources of medicine throughout history. The medicinal properties of plants are being investigated in recent scientific research throughout the world due to their potent antioxidant activities, no side effects and their economic viability (Joyce 1987).
Pistacia integerrima (Anacardiacea) leaf galls, commonly known as kakadshingi, is an appendage of the plant. Its medicinal uses are diverse and include its use in coughs, asthma, fever and respiratory disorders. It is reported to be useful in liver disorders (Anonymous 1976). The leaf galls are used as a tonic and stimulant. Galls are used in the form of a decoction or lotion as a gargle and mouthwash to suppress hemorrhage from gums. It is also used to suppress bleeding from the nose. Hakim consider galls useful in pulmonary infections, diarrhea and vomiting (Nadkarni 1976). Pistacia integerrima showed the presence of gallic acid, quercetin, leuteolin and chebulenic acid (Hirori 1966, Kalidhar 1985). The preliminary investigation on extracts has shown significant hepatoprotective activity (Lubuncic 2005). The present study aims to validate the claims made on the plant using in vitro hepatocytes.
Isolated hepatocytes have become a useful model for pharmacological, toxicological, metabolic and transport studies on xenobiotics since the development of techniques for high yield isolation of rat hepatocytes (Roberfroid 1991). The present study was undertaken to standardise an in vitro test system using primary cultured rat hepatocytes to detect the protective effect of extracts against paracetamol induced cellular damage, as the in vitro model can offer a more detailed approach to understanding the mechanism of toxic, thus hepatoprotective action of drugs being developed (Olmos 2007). In the present study compounds isolated from the ethyl acetate fraction of methanol extract were examined against the hepatotoxin in vitro using primary cultured rat hepatocytes. Silymarin was used as the positive control.
Materials and methods
Pistacia integerrima leaf galls were purchased from the local market of Pune, India and authenticated at Regional Research Institute (Ayurveda), Pune.
Chemicals: Nitroblue tetrazolium (NBT), EDTA, sodium nitropruside, thiobarbituric acid, L ascorbic acid were purchased from Qualigens, India. DPPH was purchased from Sigma Aldrich, US. All the chemicals and solvents used were of analytical grade.
Animals: Albino rats of either sex (100-150 g) were obtained from M/S Zydus Research Center, Ahmedabad and were housed under standard environmental conditions with free access to food and water. The experiments were performed after the experimental protocol approval from Institutional Animal Ethics Committee, M. S. University, Baroda Vadodara, Gujrat, India.
Isolation of compounds (Kalidhar 1985)
Approximately 23 g of ethyl acetate fraction of methanol extract of P. integerrima was obtained by fractionation of approximately 50 g of methanolic extract. This extract was further subjected to column chromatography using silica gel of 60-120 mesh size and eluted with hexane: ethyl acetate (1:1) and ethyl acetate: chloroform (7:3). The compounds isolated were subjected to HPTLC to confirm the singularity. The percentage purity of the isolated compounds was assessed by HPLC. The isolated compounds were quantified in the methanol fraction by HPTLC. The compounds were subjected to physicochemical parameters and spectral studies.
Assessment of the percentage purity of isolated compounds by HPLC
The isolated compounds were subjected to HPLC for determination of percentage purity. Reagents: Acetonitrile, methanol, water (HPLC grade). Preparation of sample: Sample solutions were prepared by dissolving 1 mg/mL of isolated compounds in methanol. Further dilution was made in mobile phase i.e. acetonitrile:methanol:water (80:15:5). Chromatographic conditions: The chromatographic separation was achieved on [C.sub.18] Phenomenex column (250mm x 4.6mm, 5 [micro]m). The mobile phase was filtered through nylon filter 0.2 [micro]m and degassed before use. The flow rate was 1 mL/min.
Quantification of isolated compounds
Sample preparation: P1 and P2 were evaluated quantitatively in methanolic extract of P. integerrima. 1 mg/mL sample solution was prepared. Preparation of standard stock solution: standard stock solution containing quercetin and gallic acid 1 mg/mL was prepared in methanol. Mobile phase: ethyl acetate:methanol:water (100:13.5:10) was used as mobile phase for quercetin and toluene:ethyl acetate:formic acid:methanol (3:3:0.8:0.2) was for gallic acid. HPTLC instrumentation: a Camag HPTLC system equipped with an automatic TLC sampler, TLC scanner 3, UV cabinet and twin trough glass tank was used for analysis (Jegannath 2008, Swaroop 2005)
Preparation of calibration curve: A calibration curve was established using six analyte concentrations (2-14 [micro]l) representing 2-14 [micro]g of quercetin and gallic acid. Standard zones were applied by means of Linomat V automated spray on band applicator with following settings: band length 6 mm, distance between the bands 4 mm, distance from the plate side edge 8 mm and distance from the bottom of the plate 15 mm. Plate was developed in a vapor equilibrated CAMAG twin trough chamber. After development the plates were air dried for 5 min and standard zones were quantified by linear scanning at 254 nm.
In vitro antioxidant activity DPPH free radical scavenging activity
Free radical scavenging potential of P1 and P2 fractions was tested against a methanolic solution of DPPH. A solution of DPPH 0.1 mM in methanol was prepared and 1.0 mL of this solution was added to 3 mL of different concentrations (5-400 [micro]g/mL) P1 and P2 in methanol. It was incubated at room temperature for 30 min and absorbance was measured at 517 nm against the corresponding blank solution. Ascorbic acid was taken as reference. The 5 inhibition of DPPH free radical was calculated by following equation: % scavenging activity = [(Ac- As)/ Ac] x 100. Ac is absorbance of control reaction, As is the sample. The antioxidant activity is expressed as IC 50 (Sjarma 2008).
Reducing power assay
The different concentration of P1 and P2 (5-60 [micro]g/mL) in 1 mL deionized water were mixed with phosphate buffer (2.5 mL, 0.2M, pH 6.6) and 1% potassium ferricyanide (2.5 mL). The mixture was incubated at 50[degrees]C for 20 min. Trichloro acetic acid (2.5 mL 10%) was added and mixed with distilled water (2.5 mL) and ferric chloride (0.5 mL, 0.1%) and absorbance was measured at 700 nm. Ascorbic acid was taken as standard (Rucj 1989).
Scavenging of hydrogen peroxide
A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). The concentration of hydrogen peroxide was determined by recording the absorbance at 230 nm. Different concentrations of fractions (5-100 [micro]g/mL) in distilled water were added to hydrogen peroxide solution (0.6 mL, 40 mM). The absorbance was measured at 230 nm after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging was calculated by % scavenging activity = [(Ac- As)/ Ac] x 100. Ac is absorbance of control reaction, As is absorbance of sample (Halliwell 1987).
Hydroxyl radical scavenging activity
The assay was performed by adding 0.1 mL EDTA, 0.1 mL hydrogen peroxide, 0.36 mL deoxyribose, 1 mL test solution (10-100[micro]g/mL) dissolved in distilled water, 0.33 mL, of phosphate buffer (50 mM, pH 7.4) and 0.1 mL ascorbic acid in sequence. The mixture was incubated at 37[degrees]C for 1 hr. A 1 mL portion of the incubated mixture was mixed with 1 mL of 10% trichloroacetic acid and 1.0 mL of 0.5% thiobarbituric acid to develop pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity was reported as percentage inhibition of deoxyribose degradation and calculated as % scavenging activity = [(Ac- As)/ Ac] x 100. Ac is absorbance of control reaction, As is absorbance of sample (Halliwell 1987).
In vitro hepatoprotective activity Hepatotoxins and test substances
Paracetamol 300 [micro]g/mL was used to produce submaximal toxicity in isolated rat hepatocytes. The test solutions were tested at dose level 100, 500 and 1000 [micro]g/mL. Silymarine was used as positive control at dose level 100 [micro]g/mL. All the solutions were dissolved in 30% DMSO.
Isolation of rat hepatocytes
Hepatocytes were isolated from rat liver as per the reported method by Sarkar and Sil with some modifications (Sarkar 2005). The liver was isolated under aseptic conditions and placed in chilled HEPES (N-2-hydroxyethylpiperazine-N-2 ethane sulphonic acid) buffer containing HEPES (0.01M), NaCl (0.142M) and KCl (0.0067M), pH7.4. The liver pieces then incubated in a second buffer containing HEPES (0.01M), Nacl (0.142M) and KCl (0.0067M) and collagenase type IV, at pH 7.6 for about 45 min at 37[degrees]C. Hepatocytes were obtained after filtration through muslin cloth and cold centrifugation (4[degrees]C, 200 rpm/min for 2 min three times) and resuspended in 4-5 ml HEPES buffer I. The viability of the hepatocytes was assessed by trypan blue exclusion method (Kiso 1983).
Primary cultures of rat hepatocytes
The method of Tinstorm and Obrink (1989) with significant modification was used for this purpose. The freshly isolated viable hepatocytes were suspended in the culture medium RPMI-1640 supplemented with calf serum (10%), HEPES and Gentamycin (1[micro]g/mL). These cells approximately 1.2 x [10.sup.6]/mL were seeded into culture bottles and incubated at 37[degrees]C in atmosphere of 5% C[O.sub.2]. The hepatocytes formed a monolayer upon incubation for 24 hrs. The newly formed cells were round and mostly appeared as individual cells. These cells were 96-97% viable as confirmed by trypan blue.
Hepatic cytotoxic testing
The isolated compounds P1 and P2 were tested for their hepatic toxicity at 10, 50 and 100 [micro]g/mL on isolated rat hepatocytes. After 24 hrs incubation at 37[degrees]C in C[O.sub.2] incubator, percentage viability of hepatocytes was tested by Trypan blue exclusion method and by estimation of total protein content.
Twenty four hours after the establishment of the monolayer of the hepatocytes, the medium was decanted and the culture was washed with HEPES buffer I and finally the hepatocytes were suspended in 5mL of HEPES buffer I. The hepatic toxicity was induced with paracetamol 300 [micro]g/mL. Test substances including silymarine were dissolved in 30% DMSO. Hepatocytes suspension (0.1 mL) in triplicate were distributed into various culture plates labeled as control, toxicant, standard (silymarine + toxicants) and test (test samples plus toxicants).
The control group received 0.1 mL of vehicle (30% DMSO) and toxicant group received 0.01 mL of respective test solutions (100, 200 and 500 [micro]g/mL of extracts/ fractions dissolved in 30% DMSO) followed by 0.1 mL of silymarine solution (100 [micro]g/mL) followed by respective hepatotoxin. The contents of all the tubes were made up to 1 mL with HEPES buffer I. The contents of all the plates were mixed well and incubated for 24 hrs at 37[degrees]C. The test and standard groups of hepatocytes were incubated with respective solution for 30 min and then exposed to hepatotoxin. After incubation hepatocytes suspensions were collected to assess cell damage by trypan blue exclusion method. Hepatocytes suspensions were centrifuged at 200 rpm. The leakage of enzymes GOT, GPT and total proteins secreted outside the cells were determined from the supernatant by using standard kits for enzyme estimation (Kurma 1998).
Assessment of hepatoprotective activity
The effect of different extracts in the liver protection was determined by measuring an increase in the percentage of viable cells in the treatment group compared with the control and the toxicant groups. Reversal of toxin induced elevation in the levels of enzymes and toxin induced reduction in the levels of proteins were considered to be the important criterion of hepatoprotective activity. The UV kinetic method based on the reference method of International Federation of Clinical Chemistry (Bergmeyer 1985) in which both SGPT and SGOT were assessed on the basis of enzyme coupled system where keto acid formed by the aminotransferase reacts in a system using NADH.
The coenzyme is oxidized to NAD and the decrease in absorbance at 340 nm is measured. For SGOT malate dehydrogenate is used to reduce oxaloacetate to malate where as in SGPT the pyruvate formed in the reaction is converted to lactate by lactate dehydrogenase was followed for the assessment of activity of glutamic oxaloacetic transaminase and glutamic pyruvate transaminase in the cells. Total protein was estimated by Biuret test where protein produces a violet color complex with copper ions in the alkaline solution. The absorbance of the color complex is directly proportional to the protein in the sample.
The mean value [+ or -] were calculated for each parameter. Percentage reduction against the hepatotoxin by the test sample was calculated by considering the difference in the enzyme level between the hepatotoxin treated group and control group as 100% reduction. Each parameter was analyzed by one way ANOVA followed by Bornferronis test.
The isolated compounds P1 and P2 were confirmed as quercetin and gallic acid respectively. Their identity was confirmed by co TLC, physicochemical parameters and spectral studies. The spectra were matched with that of standard compound. The % purity of P1 and P2 was assessed by HPLC method which was found to be 83.6% and 97.5% respectively. Isolated quercetin (P1) and isolated gallic acid (P2) in the total methanol extract were quantified by HPTLC and were found to be 4.94% and 5.77 % respectively.
[FIGURE 1 OMITTED]
The isolated compounds were subjected to antioxidant activity by DPPH radical scavenging activity, reducing power activity, hydrogen peroxide radical scavenging and hydroxyl assay. In all the experiments the isolated compounds showed dose dependant antioxidant activity (Figure 1).
Hepatic cytotoxic testing was performed by treating normal cells with the isolated compounds. When the normal hepatocytes were treated with the extracts under test, there was no alteration in the value of % viable cells and TPTN content compared with control at the dose level up to 1000 [micro]g/mL indicating that the extracts were not toxic to the cells. Both P1 and P2 showed significant activity at 50 and 100 [micro]g/mL. Cell viability with silymarine was found to be 81.55%. When treated with P1 and P2 at 50 and 100 [micro]g/mL the viability was increased as compared to the toxicant. Similarly there was increase in the enzyme level GPT and GOT in the paracetamol treated group which was found to lowered in the treatment group. The results were comparable to that of silymarin (Table 1).
The present studies were performed to evaluate antioxidant and in vitro hepatoprotective activity of P. integerrima. Most mammals have an inherited mechanism to prevent and neutralise the free radical induced damage. It is apparent from the present study that quercetin and gallic acid not only scavenge the free radicals but also inhibit generation of free radicals. It has previously been reported that naturally occurring phenolic compounds have free radical scavenging properties due to their hydroxyl groups (Diplock 1997, Rice 1998). Both quercetin and gallic acid are reported to possess good antioxidant properties.
Isolated hepatocytes have become a powerful model for pharmacological, toxicological, metabolic and transport studies of xenobiotics since the development of techniques for high yield isolation of rat hepatocytes (Sureshkumar 2008). Freshly isolated rat hepatocytes are very useful and a common tool for study of cytotoxicity and metabolic studies in this area as they keep enzymatic activity similar to in vivo for several hours. Various hepatotoxins viz carbon tetrachloride, paracetamol and thioacetamide have been shown to result in reduction of viability of hepatocytes and leakage of enzymes which are considered to be the markers of cellular injury (Zimmeramann 1965).
Similar changes in the present study confirm the satisfactory standardisation of our isolation and culture procedures. Paracetamol is metabolized by microsomal cytochrome P 450. The hepatotoxicity of paracetamol is due to formation of toxic and highly reactive metabolite N acetyl p benzoquinoneamine. This highly toxic substance starts a chain of free radicals which attack membrane lipids and proteins thereby causing destruction of microsomes and liver cells leading to cell lysis. Leakages of cytosolic enzymes out of the cells thus occurs due to an increase in cell permeability, membrane damage and cell necrosis (Recknagael 1967, Germano 1999).
In the present study it was observed that there was reduction in cell viability due to injury to plasma membrane. The enzyme level was increased due to leakage which was restored with the treatment with quercetin and gallic acid. Incubation of hepatocytes with quercetin and gallic acid increased cell viability as well as altered biochemical parameters induced by hepatotoxin.
The in vivo studies require a large number of animals (n=6) and need up to 3-5 days of drug administration for a significant effect to be produced, using large amounts of drugs. On the other hand the in vitro model is rapid and requires fewer amounts of test substances. Bioactive fractions obtained from the plant extracts are usually available in the small quantities. Therefore in vitro models can be more useful in assessment of activity.
In the literature many authors have reported hepatoprotective activity of phenolic and flavonoidal compounds. Galisto et al (2006) reported the hepatoprotective activity of flavonoids of Rosmarinus tomentosus. The hepatoprotective effect of quercetin and rutin was reported by Janbaz (2002, 2004). Silymarin from Silybum marianum is a good hepatoprotective agent (Hiroshi 1998).
In accordance with these findings it may be hypothesised that hepatoprotective activity of P. integerrima may be attributed to quercetin and gallic acid. In conclusion the study confirms the therapeutic potential of P. integerrima.
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* Joshi Uttari P, Mishra SH
Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda
* Corresponding author email: firstname.lastname@example.org
Table 1 Effect of P. integerrima against paracetamol induced toxicity in rats Group Viable cells (%) Control 97.03 [+ or -] 0.26 Paracetamol (300 [micro]g/mL) 27.65 [+ or -] 1.43 Sylimarine (100 [micro]g/mL) 84.23 [+ or -] 1.26 * (81.55) P 1 (10 [micro]g/mL) 84.23 [+ or -] 1.26 * (81.55) P 1 (50 [micro]g/mL) 49.74 [+ or -] 1.19 * (58.64) P 1 (100 [micro]g/mL) 56.34 [+ or -] 1.22 ** (41.35) P 2 (10 [micro]g/mL) 51.26 [+ or -] 0.91 * (34.04) P 2 (50 [micro]g/mL) 60.21 [+ or -] 2.72 * (46.92) P 2 (100 [micro]g/mL) 72.26 [+ or -] 3.01 * (64.29) Group GOT (IU/L) Control 20.35 [+ or -] 0.53 Paracetamol (300 [micro]g/mL) 46.32 [+ or -] 0.35 Sylimarine (100 [micro]g/mL) 22.86 [+ or -] 0.81 * (90.00) P 1 (10 [micro]g/mL) 22.86 [+ or -] 0.81 * (90.00) P 1 (50 [micro]g/mL) 38.51 [+ or -] 0.59 * (28.19) P 1 (100 [micro]g/mL) 28.23 [+ or -] 0.64 ** (68.44) P 2 (10 [micro]g/mL) 45.26 [+ or -] 1.31 (15.0) P 2 (50 [micro]g/mL) 33.96 [+ or -] .036 ** (46.18) P 2 (100 [micro]g/mL) 29.51 [+ or -] 0.83 * (63.98) Group GPT (IU/L) Control 24.63 [+ or -] 0.27 Paracetamol (300 [micro]g/mL) 55.62 [+ or -] 0.69 Sylimarine (100 [micro]g/mL) 29.06 [+ or -] 0.48 * (85.70) P 1 (10 [micro]g/mL) 29.06 [+ or -] 0.48 * (85.70) P 1 (50 [micro]g/mL) 43.26 [+ or -] 0.87 * (39.88) P 1 (100 [micro]g/mL) 34.25 [+ or -] 0.54 * (68.15) P 2 (10 [micro]g/mL) 43.02 [+ or -] 1.09 * (40.65) P 2 (50 [micro]g/mL) 35.21 [+ or -] 1.07 * (65.85) P 2 (100 [micro]g/mL) 31.25 [+ or -] 1.89 ** (78.63) Group TPTM (g/dL) Control 4.16 [+ or -] 0.12 Paracetamol (300 [micro]g/mL) 2.09 [+ or -] 0.51 Sylimarine (100 [micro]g/mL) 3.68 [+ or -] 0.26 * (76.81) P 1 (10 [micro]g/mL) 3.68 [+ or -] 0.26 * (76.81) P 1 (50 [micro]g/mL) 3.01 [+ or -] 0.39 * (44.44) P 1 (100 [micro]g/mL) 3.20 [+ or -] 0.64 ** (53.62) P 2 (10 [micro]g/mL) 2.03 [+ or -] 0.68 (6.0) P 2 (50 [micro]g/mL) 2.69 [+ or -] 0.94 * (28.98) P 2 (100 [micro]g/mL) 3.28 [+ or -] 0.94 ** (57.48) n=3 All groups were compared again control. * p<0.5, ** p<0.01, *** p<0.001 The statistical analysis was done by one way ANOVE followed by Bornferronis test. The values in the parenthesis indicate percentage protection against toxicant.